1. Field of the Invention
The present invention relates to vitamin C derivatives linked with a peptide(s) or pharmaceutically acceptable salts thereof, a method of preparing the same, and a composition comprising the same.
2. Description of the Prior Art
The skin is the largest organ of the body, and has several different functions. In detail, the skin protects the body from bacterial invasion and chemicals in the outside world, provides a mechanical barrier to protect the body, regulates body temperature and eliminates waste products, and serves as an organ of respiration. The skin has three layers: the epidermis, the dermis and the subcutaneous tissues. The epidermis, the outer part of the skin, is the thinnest layer of the skin and includes keratinocytes and melanocytes, which are closely associated with each other. The dermis, which is the inner layer of the skin, occupies about 95% of the skin, and is responsible for regulating the moisture levels of the skin and protecting the skin. The dermis comprises a tight, sturdy mesh of collagen and elastin fibers. Both collagen and elastin are the representative proteins playing a critical role in skin resilience (wrinkling of the skin). Also, the dermis is the site where blood vessels and nerves are located, and contains fat cells and the natural moisturizer, Na-PCA or hyaluronic acid. The subcutaneous tissue, which is the innermost layer of the skin, functions to supply nutrients to the epidermis and the dermis, determine body shape, control body temperature, absorbs external impacts, protect cells under the subcutaneous tissue, etc.
However, the normal skin functions are rapidly lost with time by intrinsic or environmental factors, causing the skin to appear as aged. As skin aging proceeds, the skin's constituents, the epidermis, the dermis and the subcutaneous tissue become thin while the collagen and elastin fibers become slender and less elastic, leading to a reduction in skin resilience and eventually causing generation of wrinkles. In addition, as the lipid barrier, which serves as a skin barrier, undergoes a change in lipid content and composition, the skin has physiological changes including a reduced moisture level and increased dryness. Further, chloasma, freckles, pigmentation and other skin lesions are caused with skin aging.
In order to solve these problems associated with skin aging, many studies are performed to find or develop compounds having skin improving effects and compositions containing the compounds as effective ingredients.
Vitamin C, vitamin E, beta-carotene and other antioxidants are widely used for inhibiting skin aging. Of them, vitamin C (ascorbic acid) has an antioxidant effect as well as other various effects of protecting the skin from ultraviolet (UV) exposure, reducing wrinkling of the skin by stimulating collagen synthesis, lightening pigmentation such as chloasma, freckles and black spots, and reinforcing the immune system. The functions of vitamin C will be described in detail, below.
Vitamin C protects the skin from UV radiation, in particular, ultraviolet A (UVA) radiation (Darr, D. et al., 1996, Acta Derm. Venereol. (Strckh). 76: 264-268; Black, H. S. et al., 1975, J. Invest. Dermatol. 65: 412-414). Also, vitamin C functions to protect the skin from damage caused by ultraviolet B (UVB) radiation. When vitamin C is applied to some regions of the skin of pig and human subjects before UVB radiation, the redness (erythema) and sun burn are prevented (Darr, D. et al., 1992, Brit. J. Dermatil. 127: 247-253; Murry, J. et al., 1991, J. Invest. Dermatol. 96: 587).
In the skin, blood and other tissues, vitamin C serves as a potent in vivo antioxidant that neutralizes reactive oxygen species (ROS) generated by chemical contamination, smoking, or, in particular, UV light. This action of vitamin C is possible due to its chemical structure of easily oxidizing to dehydro-L-ascorbic acid by accepting two electrons. Vitamin C is a key element of the non-enzymatic antioxidant protection system of the skin. At high concentrations, vitamin C functions to neutralize ROS, such as singlet oxygen, superoxide anion and hydroxy radicals, before they oxidize or denature proteins, nucleic acids, membrane lipids and other biological components (Buettner, G. R. et al. 1996. Cadenas, E., Packer, L., eds. Handbook of antioxidants. pp. 91-115).
When transdermally applied to the stratum corneum of the skin, vitamin C was demonstrated to have skin appearance benefits including enhancements in luster, tone and elasticity, and reductions in wrinkles, etc. by stimulating collagen synthesis (U.S. Pat. No. 4,983,382). Biosynthesis of hydroxyproline amounting to about 10% of amino acid residues of a collagen polypeptide is stimulated by proline hydroxylase. Vitamin C acts as a cofactor of the proline hydroxylase enzyme (Tomita, Y. et al., 1980, J. Invest. Dermatol. 75(5): 379-382). That is, vitamin C has skin appearance-improving effects such as anti-wrinkling by stimulating synthesis of hydroxyproline via stimulation of the activity of proline hydroxylase and finally increasing biosynthesis of collagen with a triple helix structure.
Vitamin C has a whitening effect by inhibiting tyrosinase known to play a critical role in melanin synthesis and eventually reducing melanin production (Tomita, Y. et al., 1980, J. Invset. Dermatol. 75(5); 379-382).
As described above, vitamin C reinforces the immune system. In particular, vitamin C was proven to inhibit the release of histamine from cellular membranes, thereby reducing allergic responses on sensitive skin, and protects mice from UV-induced immunosuppression and tolerance upon skin contact with an antigen (Nakamura, T. et al., 1997, J. Invest. Dermatol. 109: 20-24). In addition, vitamin C inhibits viral infections by stimulating phagocytosis by leukocytes and leukocyte migration during viral infection, and increases the biosynthesis of interferon as a substance inhibiting the viral proliferation. In addition to the functions as noted above, vitamin C participates in the metabolism of folic acid and amino acids.
Vitamin C, which has the diverse functions as described above, is a water-soluble substance having a chemical formula of C6H8O6. The hydrophilicity of vitamin C is attributed to hydroxyl groups at carbon positions 2, 3, 5 and 6 of the molecule. At a neutral pH, such as in water, these hydroxyl groups, particularly the groups at carbon positions 2 and 3, carry negative charges. The negative charges allow vitamin C to be dissolved quickly and completely in an aqueous solution, but greatly limit the solubility of the molecule in a non-aqueous, organic environment such as the skin. Also, vitamin C at neutral pH also does not dissolve well in organic solvents commonly used in formulations for topical use, such as glycerin, propylene glycol, and various fats, thus limiting the usefulness of organic solvents as a vehicle for carrying vitamin C into the skin. That is, when vitamin C is not ionized, its absorption into the skin barrier can be easily achieved. This non-ionic state occurs only at a pH of less than 4.2.
When vitamin C is topically applied, its accumulation in the skin proved to be 20 to 40-fold higher than the cases of being orally administered. When vitamin C is topically applied, with aims of utilizing its antioxidant effect, protecting the skin from UV exposure, improving wrinkling of the skin by stimulation of collagen synthesis, lightening pigmentation such as chloasma, freckles and black spots, and reinforcing the immune system, the effective substance should pass through the outermost stratum corneum of the skin and arrive the place where cells exist in the epidermis, and, for this, should have a high percutaneous absorption rate. Typically, the percutaneous permeability of a certain substance is related with the lipophilicity of the substance. When the substance has a lipophilicity similar to the skin, it has a higher distribution coefficient into the skin, and thus, easily absorbed through the skin. However, due to its high hydrophilicity, vitamin C has a low distribution into the skin, and thus, is difficult to be percutaneously absorbed.
Many attempts were made to improve the instability and the low percutaneous absorption of vitamin C having diverse effects as described above.
Derivatives of vitamin C are classified into three types, as described below. One type includes phosphorylated ascorbic acid or metal salts thereof. Ester linkage of a hydroxyl group at a carbon position 2 or 3 of ascorbic acid and a phosphate group results in production of ascorbyl-2-phosphate or ascorbyl-3-phosphate. These derivatives of vitamin C are, unlike other derivates, easily converted to L-ascorbic acid, the only form of vitamin C that can be used by the body, but has a significant problem of being difficult to be absorbed into the skin due to their negative charges.
Another type of vitamin C derivatives includes fatty acid-linked forms of ascorbic acid. For example, U.S. Pat. No. 5,409,693 discloses fat soluble fatty acid esters of ascorbic acid, for example, ascorbyl palmitate, ascorbyl laurate, ascorbyl myristate and ascorbyl stearate. Of them, ascorbyl-6-palmitate is the most widely used. Ascorbyl-6-palmitate is easily absorbed into the skin, but is difficult to be converted to L-ascorbic acid. Ascorbyl-6-palmitate was reported to fail to protect mouse skin from photo-oxidation (Bissett, D. et al., 1990, Photodermatol Photoimmunol Photomed 7: 56-62). Also, when a serum containing 10 wt % of ascorbyl-6-palmitate was applied to pig skin, no significant increase in skin levels of ascorbic acid was observed (Pinnell, S. R. et al., 2001. Dermatologic Surgery. 27(2): 137-142).
The third type of vitamin C derivatives includes monosaccharide esters of ascorbic acid, for example, glycosylated, mannosylated, fructosylated, fucosylated, galactosylated, N-acetylglucosaminated, and N-acetylmuramic derivatives of ascorbic acid. However, they have not been concretely and accurately evaluated for in vivo physiological activity.
Collagen is very abundant in the skin, blood vessels, intestine, bone, etc, and makes up 70% of the dermis of the skin. Also, the epimysium surrounding the muscle is composed of collagen. Collagen, which makes up 30% of the total weight of body proteins, provides mechanical strength to the skin, strength with resistance to tearing and stretching to the connective tissue and cohesion to the tissues, and functions to maintain cell adherence and induce cell division and differentiation upon growth of organisms or wound healing. Collagen is synthesized in the fibroblasts. Its levels are reduced by intrinsic aging and photoaging, and are known to be reduced by 65% during the ages ranging from 20 to 80 (Shuster S., 1975, British Journal of Dermatology, 93(6): 639-643). Active synthesis of collagen in vivo results in an increase in the levels of dermal substrate components, which is responsible for wound healing, increased skin elasticity, reduced wrinkles, and the like. With this identification, collagen is used in cosmetic products, foods, medicinal products, etc. As a minimal active unit of collagen, an oligopeptide consisting of less than 10 amino acids serves as a messenger, a stimulator and a nerve impulse transmitter, and involved in physiological events, such as, growth control, lactation, immunity, digestion, increased blood pressure and wound healing. Some peptides are identified to have wound healing effects, as disclosed in France Pat. No. 2,668,265, U.S. Pat. No. 4,665,054, and International Pat. Publication Nos. W091/3488 and W091/7431. However, these peptides have a disadvantage of causing a great reduction in product stability due to their property to form a precipitate in a composition.